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Drosophila adult leg development provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. A pulse of the steroid hormone ecdysone at the onset of metamorphosis triggers the rapid transformation of a flat leg imaginal disc into an immature adult leg, largely through coordinated changes in cell shape. In an effort to identify links between the ecdysone signal and the cytoskeletal changes required for leg morphogenesis, we performed two large-scale genetic screens for dominant enhancers of the malformed leg phenotype associated with a mutation in the ecdysone-inducible broad early gene (br1). From a screen of >750 independent deficiency and candidate mutation stocks, we identified 17 loci on the autosomes that interact strongly with br1. In a complementary screen of approximately 112,000 F1 progeny of EMS-treated br1 animals, we recovered 26 mutations that enhance the br1 leg phenotype [E(br) mutations]. Rho1, stubbloid, blistered (DSRF), and cytoplasmic Tropomyosin were identified from these screens as br1-interacting genes. Our findings suggest that ecdysone exerts its effects on leg morphogenesis through a Rho1 signaling cascade, a proposal that is supported by genetic interaction studies between the E(br) mutations and mutations in the Rho1 signaling pathway. In addition, several E(br) mutations produce unexpected defects in midembryonic morphogenetic movements. Coupled with recent evidence implicating ecdysone signaling in these embryonic morphogenetic events, our results suggest that a common ecdysone-dependent, Rho1-mediated regulatory pathway controls morphogenesis during the two major transitions in the life cycle, embryogenesis and metamorphosis.

Studies of Drosophila metamorphosis have been hampered by our inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, we have expressed GFP in specific tissues of living prepupae and pupae and compiled images of these animals into time-lapse movies. These studies reveal, for the first time, the dynamics and coordination of morphogenetic movements that could only be inferred from earlier studies of dissected staged animals. We also identify responses that have not been described previously. These include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp(5) mutants of the Broad-Complex (BR-C), and imaginal disc elongation and eversion is abolished in br(5) mutants of the BR-C. Leg malformations associated with the crol(3) mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals., (Copyright 2003 Elsevier Science (USA))

Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad (br) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Here, we use functional studies of amorphic br5 mutants and a transcriptional target approach to identify processes driven by br and its regulatory targets in leg imaginal discs. br5 mutants fail to properly remodel their basal extracellular matrix (ECM) between 4 and 7 hr after puparium formation. Additionally, br5 mutant discs do not undergo the cell shape changes necessary for leg elongation and fail to elongate normally when exposed to the protease trypsin. RNA-sequencing of wild-type and br5 mutant leg discs identified 717 genes differentially regulated by br, including a large number of genes involved in glycolysis, and genes that encode proteins that interact with the ECM. RNA interference-based functional studies reveal that several of these genes are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, br Z2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, our results suggest that br Z2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the midprepupal stage, but must be switched off to allow expression of pupation genes. [ABSTRACT FROM AUTHOR]